Rapid evolution of A(H5N1) influenza viruses after intercontinental spread to North America

Highly pathogenic avian influenza A(H5N1) viruses of clade 2.3.4.4b underwent an explosive geographic expansion in 2021 among wild birds and domestic poultry across Asia, Europe, and Africa. By the end of 2021, 2.3.4.4b viruses were detected in North America, signifying further intercontinental spread. Here we show that the western movement of clade 2.3.4.4b was quickly followed by reassortment with viruses circulating in wild birds in North America, resulting in the acquisition of different combinations of ribonucleoprotein genes. These reassortant A(H5N1) viruses are genotypically and phenotypically diverse, with many causing severe disease with dramatic neurologic involvement in mammals. The proclivity of the current A(H5N1) 2.3.4.4b virus lineage to reassort and target the central nervous system warrants concerted planning to combat the spread and evolution of the virus within the continent and to mitigate the impact of a potential influenza pandemic that could originate from similar A(H5N1) reassortants.

outbreaks of A(H5N6) in China which have been associated with human infections. Of note, A(H5N6) viruses of clade 2.3.4.4h had been the previously predominant clade in poultry in China and had also caused human infections 4 .
In December 2021, A(H5N1) viruses were detected in poultry and a gull in Eastern Canada. The virus responsible was closely related to 2.3.4.4b viruses identified in Europe in the spring of that year 5 . This was only the second time that a GsGD H5-lineage virus was detected in birds in the Americas; the first such occurrence was in 2014, when the infected birds had crossed the Bering Strait and entered the Pacific flyway. After the initial 2021 detection in Newfoundland, infected wild birds were reported in the U.S. south atlantic states 6 and in several other U.S. regions soon thereafter. The purpose of this study was to understand the course of the genetic and phenotypic evolution of the 2.3.4.4b viruses as they spread throughout North America (NAm). We demonstrate that westward intercontinental 2.3.4.4b expansion resulted in reassortment with NAm wild bird viruses yielding reassortants with diverse ribonucleoprotein gene mixtures. The resulting viruses have distinct in vitro phenotypes including increased virus replication rates and pH of activation, but most concerningly, they cause severe disease outcomes with dramatic neurologic involvement in mammalian animal models.

Virus pathogenesis and transmission in chickens and ferrets
We first conducted a risk assessment to determine the pathogenic and transmission properties of two early isolates in birds and mammals; A/ American wigeon/South Carolina/22-000345-001/2021 (Wigeon/SC/ 21) and A/bald eagle/Florida/W22-134-OP/2022 (Eagle/FL/22) were collected in December 2021 and February 2022, respectively (Table 1). White leghorn chickens and ferrets were initially used, both of which are susceptible to a variety of influenza viruses and are critical animal models among influenza risk assessment pipelines (IRAT, TIPRA) 7,8 . Both chicken-to-chicken and chicken-to-ferret transmission were examined. Naïve chickens were placed in the same cage as inoculated birds, and ferrets were housed in a separate cage 12 inches away from inoculated chickens (Supplementary Fig 1A). Both viruses replicated robustly in inoculated chickens but transmitted inefficiently to naïve contact chickens ( Supplementary Fig. 1B). Chickens (n = 3 per virus) directly inoculated with either Wigeon/SC/21 or Eagle/FL/22 died within 48 h post-inoculation (hpi). Two of twelve contact chickens paired with Wigeon/SC/21 inoculated chickens shed virus and met euthanasia endpoints at 4 or 7 dpi, while two additional non-shedding contacts died at 4 or 10 dpi. Three of twelve contact chickens paired with Eagle/FL/22 inoculated chickens shed virus and met euthanasia endpoints 3-4 days post-inoculation (dpi); virus was transiently detected in a fourth bird which survived ( Supplementary Fig. 1B). No aerosol transmission was detected from infected chickens to naïve ferrets ( Supplementary Fig. 1C), suggesting that the risk of transmission at the avian-mammalian interface remains low. We next assessed ferret-to-ferret contact transmission by placing inoculated ferrets in the same cage as naive ferrets. Neither virus transmitted from inoculated ferrets to naïve direct-contact ferrets (Fig. 1A, and Supplementary  Table 4). Surprisingly, there were dramatic differences in disease severity among directly inoculated ferrets between the two viruses. Consistent with a recent report 9 , Wigeon/SC/21 infection resulted in mild disease and the ferrets survived (Fig. 1B). Only one of three Wigeon/SC/21 inoculated ferrets exhibited weight loss (Fig. 1C), and upper respiratory tract virus shedding was detected until 5 or 7 (n = 1 ferret) dpi (Fig. 1D). In contrast, Eagle/FL/22 inoculation resulted in rapid weight loss (Fig. 1C), lethargy, and severe neurologic symptoms, including ataxia and hindlimb paralysis (Supplementary Table 4). By 7 dpi, all Eagle/FL/22 inoculated ferrets reached humane endpoints (Fig. 1B).

Genotypic and antigenic analyses of clade 2.3.4.4b viruses
To begin to understand molecular determinants contributing to the higher virulence of Eagle/FL/22, sequence analysis was performed on A(H5N1) wild bird viruses from this outbreak, which included Wigeon/ SC/21 and Eagle/FL/22. These data revealed that reassortment event(s) among H5 clade 2.3.4.4b viruses and NAm wild bird influenza viruses had occurred soon after introduction of the A(H5N1) viruses into NAm (Fig. 2). Whereas Wigeon/SC/21 had the same genotype as the first viruses detected in Canada and those detected in Europe in the spring of 2021, Eagle/FL/22 had undergone reassortment, and acquired NAm wild bird-lineage polymerase basic 2 (PB2), polymerase basic 1 (PB1), and nucleoprotein (NP) genes ( Fig. 2A).
In total, we identified four different viral genotypes among the 58 A(H5N1) viruses sequenced in this study (Fig. 2B). All viruses maintained the parental Eurasian-origin (EA) HA, NA, matrix (M), and nonstructural (NS) gene segments, but they had different combinations of polymerase and NP gene segments of either EA or NAm origin. In all cases except for PB2, for which two phylogenetically distinct genes were detected ( Fig. 2A), the NAm gene segments were monophyletic, suggesting that a single or minimal number of reassortment events had occurred, with the resulting viruses spreading geographically. The NAm-lineage proteins of A(H5N1) viruses had no markers associated with increased virulence in mammalian hosts 10,11 , and all viruses remained antigenically homogeneous, as determined by hemagglutination inhibition (HAI) assays (Supplementary Table 1).

Pathotyping of additional clade 2.3.4.4b viruses in ferrets
To further assess if virulence was linked to acquisition of NAm gene segments, we used the ferret model to pathotype four additional A(H5N1) viruses of genotypic diversity represented later in the outbreak period (Fig. 3A). These viruses were A/Fancy Chicken/  3D). All EA/NAm viruses were detected in multiple organs confirming systemic viral spread, while the native constellation virus Ck/ NL/21 had very low or trace amounts of virus outside the respiratory tract (Fig. 3E). Additionally, infection with the EA/NAm viruses resulted in higher temperature increases than Ck/NL/21 along with respiratory and neurologic symptoms (Supplementary Table 4).

Pathology
Virulence trends were also reflected in the histopathologic examina- Virus Pathogenesis in the mouse model   . P values were calculated using two-way ANOVA with Tukey's multiple-comparison post hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. these viruses induced neurologic symptoms in mice, along with weight loss and virus replication in the lungs and brains. Viruses causing moderate or no lethality in ferrets, including Eagle/NC/2, Ck/ NL/21, and Wigeon/SC/21, had the highest mouse LD 50 s at >6 log 10 EID 50 /mL (Supplementary Figs. 6 and 7).

Phenotypic properties of clade 2.3.4.4b viruses
Several influenza virus characteristics are known to contribute towards mammalian infection and spread, including receptor binding properties, pH of HA activation, and polymerase activity [12][13][14][15][16][17] . Wigeon/SC/21 and Eagle/FL/22 bound strongly to sialic acid receptors with a 3′SLN linkage to the underlying sugar (preferred by avian viruses), but poorly to 6′ SLN-linked sialic acid receptors (preferred by human viruses) (Fig. 4A, B). The pH of HA activation and inactivation segregates with host adaptation 17 , and the pH of HA activation for A(H5N1) viruses in avian species is 5.6-6.0 12 . Consistent with this, both Wigeon/SC/21 and Eagle/ FL/22 had a relatively high pH of HA activation of 5.8 by syncytium assay (Fig. 4C and Supplementary Fig. 7). These values were slightly higher than the pH of HA activation of the control human virus A/California/ 04/2009 (CA/04) (H1N1)pdm09 (pH 5.6) and substantially higher than the pH of HA activation of most human-adapted influenza viruses 14 4C). A virus inactivation pH that is lower than the HA activation pH is rare but has been observed with some swine H1 and H3 isolates and a bat A(H9N2) isolate 18 . The polymerase activities of Wigeon/SC/21 and Eagle/FL/22 were not statistically different when measured in the reporter gene assay with transiently expressed proteins (Fig. 4D). However, we detected differences in the replication rates of whole virus in non-differentiated Calu-3 cells (Fig. 4E) and primary differentiated human airway cultures (Fig. 4F) Protective antibody immunity to influenza virus targets the HA and NA and is an important consideration when assessing the risk posed by zoonotic influenza viruses 16 . Using a set of 48 human serum samples obtained from blood donors aged 18 to 46 years, we examined the presence of such cross-reactive antibodies. As expected, there were generally no significant neutralizing titers (serum dilution >1:20) to H5 HA protein (Fig. 4G). In contrast, an enzyme-linked lectin assay (ELLA) targeting antibodies to the N1 protein revealed the geometric mean titers (GMTs) of antibodies against both Wigeon/SC/21 and Eagle/FL/22 to be equivalent to those of antibodies against the seasonal CA/04 (H1N1)pdm09 NA in this serum panel (Fig. 4H). The NA proteins of the A(H5N1) viruses of clade 2.3.4.4b and those of A(H1N1) pdm09 viruses have 89.6% amino acid identity, with considerable conservation at some antigenic sites 19,20 .

Antiviral susceptibility of clade 2.3.4.4b viruses
To evaluate whether the available antiviral therapies would be effective against the A(H5N1) clade 2.3.4.4b viruses, we examined the phenotypic susceptibility of these viruses to antivirals from two U.S. FDA-approved classes: the NA inhibitors (NAIs) oseltamivir and zanamivir and the active metabolite of the PA endonuclease inhibitor baloxavir marboxil. All six viruses tested (both reassortant and native constellations) had IC 50 or EC 50 values comparable to those of drugsusceptible human A(H1N1)pdm09 influenza reference viruses (Table 2). Additionally, genotypic M gene analysis revealed the absence of amino acid changes associated with reduced susceptibility to the adamantane class of drugs (Table 2).

Discussion
With the spread of A(H5N1) viruses throughout the United States and the detection of an infection in a human 21 , the increased virulence of the reassortant viruses is of considerable concern. Except for pockets of endemic clade activity in South and Southeast Asia, clade 2. 3 3 . From a public health perspective, the increased pathogenicity of the reassortant A(H5N1) viruses is of significant concern. However, this is tempered by the avian virus-like characteristics of the viruses with respect to their receptor binding preference and their pH of HA activation. Modification of these characteristics are likely required to enable sustained human-tohuman transmission, although only a few amino acid changes among various influenza proteins are needed to switch these properties during adaptation in mammals 22,23 . In addition to the representative viruses we assayed phenotypically, we did not identify any mutations previously associated with 6′SLN-linked sialic acid binding or alterations in pH of HA activation in the viruses we sequenced. To date, a single case of human A(H5N1) infection in an individual involved in poultry culling has been detected in North America during the current outbreak 21 . This individual underwent isolation and oseltamivir treatment and recovered after experiencing only minor symptoms. Publicly deposited sequence from samples collected from this case (albeit partial and missing PB2 and NP segments) were of Eurasian origin. Zoonotic transmission is likely to continue if this virus remains present in North American wild birds, and vigilance must be maintained as these birds continue their migrations. While direct translation of animal model findings to humans must be done cautiously, future zoonotic infections with the reassortant A(H5Nx) viruses may well include severe cases. Consistent with this possibility is the recent identification of a severe reassortant virus infection of a child in Ecuador 24 .
The A(H5Nx) clade 2.3.4.4 viruses originally identified in NAm in 2014-2015 ultimately disappeared from avian species for reasons that are unclear 25 , but previous experience is unlikely to predict future events in this situation. Like the current NAm clade 2.3.4.4b viruses, the 2014-2015 viruses reassorted soon after detection on the continent, but this reassortment was not associated with changes in mammalian pathogenicity 26 . In contrast, our study with contemporary 2.3.4.4b viruses revealed that reassortants containing increasing numbers of NAm gene segments exhibited enhanced virulence with neurological involvement in mammalian models, including ferrets. However, these viruses retained avian-like receptor specificity, did not possess molecular markers of mammalian adaptation, or the ability to transmit between ferrets. Clade 2.3.4.4b viruses have thus far been associated with infections within a European mink farm 27 , spread among aquatic mammals in the Americas [28][29][30] , and reported identification from a variety of other NAm mammals including foxes, skunks, bobcats, mountain lions, and bears 31 . The increasing prevalence of 2.3.4.4b viruses are also changing the dynamics of disease in Europe, with the potential for transition from epizootic to enzootic status 32 . Our data highlight how quickly things can change in a natural system, and the potential for further A(H5Nx) reassortment and phenotypic diversification will only increase as the unprecedented global distribution of these viruses broadens. The data are shown as the mean ± SD from duplicate wells and representing one of two independent experiments. C Kinetics of pH inactivation of the Wigeon/SC/21, Eagle/FL/22, and CA/04 (H1N1)pdm09 viruses at 37°C. The data are shown as the mean ± SD from triplicate wells representing one of three independent experiments. D Minireplicon polymerase activities of Wigeon/SC/21 and Eagle/FL/22 at 37°C. The data are shown as the mean ± SD of 3-4 measurements over the hypothesized pH range for avian viruses, and 2 measurements over previously described ranges for control virus CA/04 (H1N1)pdm09, and representing one of three independent experiments. NS = not significant as determined by paired, two-tailed t-test. E Viral replication kinetics in Calu-3 cells. Cells were inoculated at an MOI of 0.001 and incubated at 37°C. The data are shown as the mean ± SD from triplicate wells and representing one of two independent experiments. F Viral replication kinetics in primary differentiated human airway cultures. Cultures were inoculated at an MOI of 0.005 and incubated at 37°C. The data are shown as the mean ± SD from triplicate culture inserts and representing one of two independent experiments. The number of inserts with viral replication out of the total is indicated in parentheses. Statistical significance (one-way ANOVA) was determined by comparison to rg-A/Texas/71/2017 (H3N2) at 72hpi. G, H Neutralizing antibody levels in human serum samples (n = 48) against HA protein (as measured by HI assay, dotted line indicates limit of detection of 1:10 serum dilution) or NA protein (as measured by ELLA assay). Points joined by lines represent values for the same individual for the individual antigens tested. * P < 0.05, *** P < 0.001, **** P < 0.0001. . Animal holding rooms were on a 12 h light/12 h dark cycle, with dry-bulb temperatures set below each species lower critical temperature to minimize potential heat stress (71°C ± 1°C with alarm points beyond this value). Room humidity was set to 45%, with active humidification of each room allowing ≥40% humidity during winter months. Human sera were purchased from a commercial provider (BioIVT, Hicksville, NY) who played no role in our study. The specimens were not collected specifically for our study, and we had no access to the subject identifiers linked to the specimens. This study utilized USDA-classified select agents and A(H5N1) viruses used herein are subject to the guidelines of, and compliance with, requirements discussed in Title 9 (CFR Parts 121 [Possession, Use, and Transfer of Select Agent Toxins] and 122 [Importation and Transportation of Controlled Organisms and Vectors]).

Influenza virus propagation and titration
The A(H5N1) influenza viruses were isolated from wild birds in the allantoic cavities of 10-day-old embryonated chicken eggs (eggs) at 35°C for up to 48 h. Seasonal influenza A(H1N1) or rg-A(H3N2) viruses were grown in MDCK cells at 37°C for up to 72 h. Viral titers were determined by injecting 0.1 mL of 10-fold dilutions of virus into the allantoic cavities of 10-day-old eggs and then calculating the 50% egg infectious dose (EID 50 ) or by inoculating MDCK monolayers and then calculating the 50% tissue culture infectious dose (TCID 50 ) by the method of Reed and Muench 33 . The lower limit of virus detection was 1.0 log 10 TCID 50 /mL or 1.0 log 10 EID 50 /mL.

Replication kinetics
Calu-3 cells (approximately 9 × 10 5 cells/well in 6-well plates) were washed with phosphate-buffered saline (PBS) and inoculated with virus (MOI of 0.001) in 2.0 mL of infection medium (MEM, 1% bovine serum albumin [BSA, Sigma-Aldrich], 0.3-1 μg/mL TPCK-trypsin [for non-A(H5N1) viruses], 1× penicillin/streptomycin/amphotericin B). One hour later, the inocula were removed, the monolayers were washed with PBS, and the supernatants were replaced with 3.0 mL of infection medium. Primary differentiated human airway cultures (MatTek, AIR-100) on trans-well inserts were cultured at an air-liquid interface using manufacturer provided medium in the basal chambers and no medium the apical chambers. Cells were washed and inoculated (MOI of 0.005), with the addition of a low pH 2.0 physiological saline wash to inactivate residual virus. No medium was added back to the apical chambers. At each timepoint, 200 µL of BEBM (Lonza) supplemented with 1% BSA was added to the apical chambers for 15 min at 37°C, then harvested. All cells or insert supernatants were sampled at 1 to 72 hpi as indicated, and virus titers (log 10 TCID 50 /mL) were determined in MDCK cells by the method of Reed and Muench 33 . Data are presented for one of two independent experiments conducted using triplicate wells/inserts for each time point and for each virus among each cell line.

Syncytium assay
Syncytium formation in vero cells was assayed as described 34 . Briefly, vero cells were seeded in a 12-well plate (1.5 × 10 5 cells/well) and incubated at 37°C overnight. The cells were then washed with PBS, inoculated with each virus at an MOI of 3.0, and incubated at 37°C for 1 h. The inocula were then removed and replaced with 1.0 mL of infection medium. At 6 hpi [for the A(H5N1) viruses] or 24 hpi [for the CA/04 (H1N1)pdm09 virus], the supernatants were removed, the cells were washed with PBS, 1.0 mL of infection medium supplemented with 5 µg/mL of TPCK-treated trypsin was added, and the plates were incubated at 37°C for 10 min. The infection medium was then removed, and residual trypsin was neutralized with 1.0 mL of MEM supplemented with 5% FBS. The supernatants were replaced with 0.5 mL of PBS, the monolayers were examined visually to verify their confluence, then the PBS was replaced with an equal volume of pHadjusted buffer (pH 5.5-6.0) for 5 min. The pH buffer supernatants were then removed, and the cells were washed with PBS and incubated in 1.0 mL of MEM supplemented with 5% FBS at 37°C for 3 h. The cells were fixed and stained using the Hema 3 Stat Pack (Fisher Scientific). Micrographs were obtained using a Nikon Eclipse TS100 microscope with a Zeiss Axiocam ERc 5 s camera at 100× total magnification. The HA activation pH was defined as the highest pH at which syncytium formation was observed. The resolution of this assay was 0.1 pH units.

Mock-inoculated cells (treated with PBS only) and cells inoculated with
CA/04 (H1N1)pdm09 virus were used as negative and positive controls, respectively.

pH of inactivation
The A(H5N1) influenza viruses were standardized to a concentration of 1.6 × 10 7 TCID 50 /mL and incubated with pH-adjusted buffer at 37°C for 1 h. Samples were neutralized by adding infection medium. The infectious titers of neutralized samples were determined in MDCK cells by TCID 50 assay 33 . The resolution of this assay was 0.2 pH units. The CA/04 (H1N1)pdm09 virus was used as a positive control.

Enzyme-linked lectin assay (ELLA)
The presence of NA-specific antibodies was determined in ELLAs 35 . Tested human sera samples were purchased from a commercial vendor (BioIVT). The vendor had no role in the study, the samples were not collected specifically for this study, and no subject identifiers linked to the specimens were available to the investigators. Briefly, flat-bottom, 96-well plates (Thermo Scientific) were coated with fetuin (Sigma-Aldrich) at 25 µg/mL in 0.1 M PBS at 4°C for 48 h. After blocking, heat-inactivated sera (inactivated at 56°C for 1 h) were serially diluted in Dulbecco PBS (DPBS) (Gibco) supplemented with 1% BSA and 0.5% Tween 20 and added to the plates. This was followed by the addition of a standardized reverse genetics (rg)-derived H6Nx antigen (virus). The plates were incubated at 37°C for 16-18 h and washed with PBS containing 0.05% Tween 20, then horseradish peroxidaseconjugated peanut agglutinin (Sigma-Aldrich) was added at 1 μg/mL and the plates were incubated for 2 h at room temperature. The plates were then washed and 3,3',5,5'-tetramethylbenzidine (Sigma-Aldrich) was added. The color reaction was stopped after 10 min by adding 1 N H 2 SO 4 . The plates were read at 450 nm for 0.1 s in a Synergy H1 microplate reader. The NI titers were defined as the reciprocal of the last dilution that resulted in at least 50% inhibition.
The fluorescence signal of the released 4-methylumbelliferone was measured using a Synergy H1 microplate reader at excitation/emission (Ex/Em) wavelengths of 360 nM/460 nM. The 50% inhibitory concentrations (IC 50 s) were estimated from dose-response curves by using the sigmoidal, four-parameter logistic non-linear regression equation

Phenotypic susceptibility to endonuclease inhibitors
The active metabolite of baloxavir marboxil, baloxavir acid (BXA), was purchased from MedChem Express. BXA susceptibility was determined by plaque-reduction assay. MDCK cells (10 6 cells/well in 6-well plates) were inoculated with a virus volume previously determined to yield approximately 50-100 plaque-forming units (PFUs). At 1 hpi, the cells were washed and overlaid with MEM containing 0.45% immunodiffusion-grade agarose (MP Biomedical), 0.1% BSA, 1 μg/mL TPCK-trypsin (for non-A(H5N1) viruses), and 10-fold dilutions of BXA (1 pM to 1 μM). At 72 hpi, the overlays were removed, and the cell monolayers were stained and fixed with 1% crystal violet, 10% formaldehyde. The number of PFU per well was calculated, and the 50% effective concentrations (EC 50 s) were determined by using the log (inhibitor) versus response logistic nonlinear regression equation (in GraphPad Prism v9). The results are representative of three independent dose-response curves and include rg-derived CA/04 A(H1N1) pdm09 viruses with the wild-type PA I38 (BXA sensitive) or the PA I38T substitution (reduced susceptibility to BXA) as reference viruses 38,39 .

Genotypic susceptibility to M2 protein inhibitors
Susceptibility to M2 protein inhibitors (amantadine, rimantadine) was determined from the M2 splice product of the full-length M gene, with analysis of the M2 amino acid substitutions (residues 26, 27, 30, 31, and 34) mediating resistance.

Generation of reverse-genetics (rg) viruses
Influenza virus gene segments were amplified from viral RNA using gene-specific primers (Integrated DNA Technologies, Supplementary

Phylogenetic analyses
Sequences other than those found in this study were retrieved from the National Center for Biotechnology Information Influenza Virus Sequence Database and from the EpiFlu database of the Global Initiative on Sharing All Influenza Data (GISAID 42 ). Sequences were then aligned, and ends were trimmed to equal lengths with BioEdit sequence alignment editor software (v.7.2.5). Similar sequences, including sequences obtained in this study, were removed, and phylogenetic relationships were inferred by the neighbor-joining method from 500 bootstrap values; topology was confirmed by the maximum likelihood method 43 ; and evolutionary analyses were conducted with the MEGA 7 software 44 .

Tanglegram
Additional North American and Eurasian influenza HA and PB2 gene sequences isolated from May 2021 to May 2022 were obtained from the GISAID database (Supplementary Table 4). Redundant PB2 sequences were eliminated by setting a sequence identity threshold of 97%, using CD-HIT (v.4.8.1) 45 . Twenty-five non-redundant PB2 sequences from the GISAID database were combined with 14 studied North American A(H5N1) sequences by using BioEdit (v.7.2.5).
Using the default parameters of Augur (v.15.0.1) 46 , the sequences were aligned, and a phylogenetic tree was constructed. Furthermore, a PB2 gene time-resolved tree was generated using TreeTime (v.0.8.6) 47 , which is included in Augur. Using exclusively the 14 studied A(H5N1) sequences, an HA gene time-resolved tree was generated through the same Augur process. These data were visualized using Auspice (v.2.29.1) 48 .

Virulence and transmission in ferrets
Four-to-six-month-old outbred influenza-seronegative male ferrets (Triple F Farms, Sayre, PA, USA) were lightly anesthetized with isoflurane and inoculated intranasally with 10 6 EID 50 units of A(H5N1) virus diluted in 1.0 mL of PBS. Ferrets (n = 3 to 6 per group) were monitored daily for clinical signs of infection. Characteristics monitored included body temperature, weight loss, relative inactivity indices 49 , ataxia, respiratory symptoms, stool consistency, and neuropathologic signs. Animals reaching the humane endpoint, according to an IACUC-approved clinical scoring system, were euthanized. Nasal washes were collected from all surviving ferrets at 1, 3, 5, 7, and 10 days post-infection (dpi). Ketamine was used to induce sneezing. At 3 and 5 dpi (in the initial phenotyping experiment) and 5 dpi (in the follow-up experiment), ferrets (n = 3 per group) were euthanized, and tissue samples were collected from the respiratory tract (nasal cavity, trachea, and lungs), the central nervous system (a combination of the brain stem, cerebellum, and frontal lobes), and the small intestine (not examined for Wigeon/SC/21 and Eagle/FL/22). Viral titers were determined in MDCK cells by TCID 50 assay. For transmission studies in the initial phenotyping experiment, three ferrets were inoculated intranasally with A(H5N1) virus as described above and housed individually in separate cages. Direct-contact transmission was assessed by placing a naïve ferret into the cage of the inoculated ferret at 1 dpi. Ferrets were monitored daily, and nasal washes were collected at 1, 3, 5, 7 and 10 dpi, starting just before the inoculated and contact ferrets were co-housed. At 25 days post contact, sera were collected from all surviving inoculated and contact ferrets and treated with receptor-destroying enzyme (Denko) as per manufacturer instructions. Degree of seroconversion was determined by hemagglutination inhibition (HAI) assay 50 , and discussed in detail in the Serological Testing section.

Pathogenicity in mice
Groups of 6-to-8-week-old female BALB/c mice (Jackson Laboratory, Bar Harbor, ME, USA) were lightly anesthetized with isoflurane and inoculated intranasally with 10-fold serial dilutions of virus suspension containing 10 2 to 10 6 EID 50 units of A(H5N1) virus in 30 µL of PBS. After virus inoculation, mice were weighed daily and monitored for mortality (death or loss of ≥25% of their body weight) and any clinical signs of infection for 14 dpi. The 50% mouse lethal dose (LD 50 ) was calculated after the 14-day observation period. For viral replication studies, BALB/c mice (n = 5 per group) were lightly anesthetized with isoflurane and inoculated intranasally with 10 4 EID 50 units of each tested virus in 30 µL of PBS. At 5 dpi, mice were euthanized, and their lungs and brains were collected and homogenized in 1 mL of infection medium. Viral titers in harvested organs were determined by TCID 50 assay in MDCK cells.

Transmission in chickens
Six-week-old specific-pathogen-free (SPF) white leghorn chickens (Charles River Laboratories, CT, USA) (n = 3 chickens per virus) were inoculated with 10 6 EID 50 units of Wigeon/SC/21 (H5N1) or Eagle/FL/22 (H5N1) virus by the natural routes (0.2 mL intranasally, 0.1 mL intraocularly, 0.1 mL intraesophageally, and 0.1 mL intratracheally). At 1 hpi, naïve chickens (n = 12 per virus) were co-housed with the donor chicken and all birds were monitored daily for disease signs. Oropharyngeal and cloacal specimens were collected at 2, 4, and 6 dpi, and the infectious viral titers were determined by EID 50 assay. To evaluate aerosol transmission from chickens to ferrets, naïve ferrets (n = 3) were housed in a ferret cage that was placed adjacent to a donor chicken cage (distance between cages was 12 inches) within the same bioisolator. Ferret nasal washes were collected at 2, 4, 6, 8, and 10 dpi, and infectious viral titers were determined by EID 50 assay. All animals were monitored twice daily for disease signs. At 21 dpi, sera were collected from all ferrets to assess the degree of seroconversion by HAI assay as described below.

Serologic testing
Sera samples ferrets or chickens were treated with receptordestroying enzyme II (Denka Seiken Co.) at 37°C overnight, heatinactivated at 56°C for 45 min. Hemagglutination inhibition (HI) titer was determined by incubating 2-fold serial dilutions of serum sample with 25 µl of 4 hemagglutinating (HA) units (HAU) in 96-well U-bottom plates (Corning). Sera and virus mixtures were incubated at room temperature for 45 min prior to addition of a 0.5% solution of chicken red blood cells (Rockland Immunochemicals) in PBS and subsequent incubation at room temperature for 30 min. The HI titers were recorded as the reciprocal of the highest serum dilution where there was complete inhibition of hemagglutination and reported as endpoint doubling dilution or Log 2 as indicated in each figure or table.

Pathology
Ferret lungs and nasal mucosa were fixed via intratracheal/intranasal infusion with 10% neutral-buffered formalin (NBF) followed by continued immersion in 10% NBF. Tissues were routinely processed and embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Serial sections were subjected to antigen retrieval for 30 min at 98°C before undergoing immunohistochemical labeling of viral antigen, using a primary goat polyclonal antibody (US Biological, Swampscott, MA) against influenza A/USSR/1977 (H1N1) virus at a dilution of 1:1000 and a secondary biotinylated donkey anti-goat antibody (Santa Cruz Biotechnology) at a dilution of 1:200.

Statistical analysis
Data were analyzed using unpaired t-tests, two-way ANOVA with Tukey's multiple-comparison post hoc test, and univariant log-rank analysis (survival curves) in GraphPad Prism v9. Replicates, group comparisons, and P values are listed in each figure legend.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.